The overall objective is to define the molecular mechanism(s) that regulates glycine and one-carbon biosynthesis in Salmonella typhimurium and Escherichia coli. Regulatory mutants will be isolated that are resistant to specific amino acid analogs. Genetic techniques (conjugation, transduction) will be used to locate the sites of these mutations on the S. typhimurium and E. coli linkage maps. Physiological studies will be performed to determine the consequences of these mutations on regulation. Strains carrying gly-lac fusions will also be used to examine regulation of the glycine pathway. A cell-free transcription-translation system will be used to examine regulation of glyA gene expression in vitro using plasmids carrying the gly-lac fusions as templates. The glyA gene from both S. typhimurium and E. coli have been cloned into plasmid vehicles. An in vitro transcription system employing restriction enzyme generated fragments as templates will be used to locate the approximate transcription start sites, and DNAase footprinting will be used to define promoter regions protected by RNA polymerase. The nucleotide sequence of the control region and glyA structural gene from both E. coli and S. typhimurium will be determined using the Maxam-Gilbert procedure.